We have previously found that docosahexaenoic acid (DHA, 22:6n-3), a highly polyunsaturated n-3 fatty acid, promotes the accumulation of phosphatidylserine (PS) and prevents apoptotic cell death in a PS- and PI3 kinase-dependent manner in neuronal cells. We have also demonstrated that n-3 fatty acid deficiency or chronic ethanol exposure markedly decreased the PS content specifically in neuronal cells where DHA is highly enriched. We have established that docosapentaenoic acid (DPA, 22:5n-6), which replaces DHA during n-3 deficiency, is not as effective as DHA in accumulating PS or preventing apoptotic cell death, thus adversely affecting neuronal survival under n-3 deficiency conditions. Similarly, long term ethanol exposure during prenatal and developmental period depletes PS from neuronal membranes through the inhibition of PS biosynthetic activities. During this period, we continued our investigation of the biochemical and signaling mechanisms underlying effects of DHA and ethanol on cell survival. We found that PS levels were uniquely modulated by the DHA status and this DHA effect was observed only in neuronal cells. The protection against apoptotic cell death induced by serum starvation was sensitive to the PS level. Substitution of DHA with DPA resulted in less accumulation of PS and accordingly less effective in supporting neuronal survival in hippocampal cultures. Prenatal ethanol exposure also decreased PS contents, especially DHA containing PS species, in various brain regions including cerebrum, cerebellum, olfactory and hippocampus. In agreement with PS-dependent survival mechanism that we have previously reported, hippocampal neurons from ethanol treated rats also showed significantly increased susceptibility to apoptosis in cultures. These data suggest that cell survival adversely affected by prenatal ethanol exposure may contribute to neurological dysfunction associated with fetal alcohol syndrome. For mechanistic understanding of our previous finding that DHA uniquely promotes hippocampal differentiation, the role of RXR, for which DHA has been shown to be an endogenous ligand, was investigated by detecting the transcriptional activation of RXR using the dual luciferase reporter assay. We found that activation of RXR required much higher DHA concentration (over 15?M) in Neuro-2A cells than 1.5 ??M at which the promotion of neurite outgrowth was observed. To test the effect of DHA directly in hippocampal primary cells, efforts are being made to improve transfection efficiency in primary cultures. As we found that Akt was a target molecule for the DHA?s antiapoptotic effect, we examined Akt conformational changes during activation using chemical cross-linking and tandem mass spectrometry. The crystal structure of full-length Akt, nor its conformational changes during activation has not been demonstrated so far. We identified in inactive Akt seven cross-linked lysine pairs. Among them, two inter-domain cross-linked pairs, K30(PH)-K389(kinase) and K284(kinase)-K426(regulatory), allowed us to monitor inter-domain Akt conformational changes during activation. Upon interacting with a model membrane containing PS/PE/PC/PIP3, these cross-linked pairs were no long observed. When Akt was activated by phosphorylation at T308 and S473, K30-K389 reappeared while K284-K426 was still missing. When the active Akt was bound to the substrate and ATP K284-K426 reappeared. These results indicated that inactive Akt exists in a folded conformation with the PH and regulatory domain covering part of the kinase domain. Upon membrane interaction, the PH and regulatory domains move away from the kinase domain, presumably exposing T308 and S473 for phosphorylation. When phosphorylated, the PH domain folds back again while the regulatory domain remains open, allowing substrate entry. The regulatory domain closes after the substrate and ATP binding. Our data provide not only the first demonstration of distinctive inter-domain conformational changes of Akt at each step of activation processes, but also a strategy for further investigations on Akt-membrane, Akt-protein and/or Akt-drug interactions in solution.